Mitogen activated protein kinases (MAPK) and nitric oxide synthases (NOSs) are critical enzymes in key signaling pathways, alterations to which impact important diseases like diabetes, atherosclerosis and cancer. MAPKs are ubiquitously expressed, very near the end of their signaling cascades and activate downstream kinases including p90 Ribosomal S6 Kinases (RSK). Together the MAPKs and other kinases including RSK are key modulators for the output of many different signals. Members of the NOS enzyme family respond to a wide variety of signals (e.g. bradykinin, VEGF, insulin) by producing nitric oxide (NO), itself an important signaling molecule. Regulation of NOS occurs through protein interactions, posttranslational modifications, cellular localization and scaffolding. MAPKs interac with their targets, including RSK and eNOS, through protein-protein interactions. The phosphorylation state of targets impacts those interactions. However, the mechanism by which MAPKs regulate NOS enzymes is unresolved. We present preliminary data to support our hypothesis that MAP kinases mediate inhibitory phosphorylations of eNOS (pS116 and/or pS602) and that activating phosphorylation at pS635 (located adjacent to the pentabasic MAP kinase docking site) by RSK (or Akt/PKA) enhance eNOS activity by inhibiting MAP kinase interaction and phosphorylation. We propose to determine if MAP kinase mediated inhibitory phosphorylations are increased in diabetic endothelial cells. Our work will determine and describe the physiological relevance of MAPK- and RSK-mediated phosphorylation of eNOS and the implications of these events for NOS activity in vitro and in primary endothelial cells. This R15 AREA project will provide critical information about the interaction of these important signaling nodes and provide a better understanding of how these connections are linked in normal and disease physiology while involving and exposing undergraduate researchers to cutting edge questions in cellular signaling, preparing them for careers in STEM fields.